The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5' UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5'UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394-405. (c)2016 AACR. [less ▲]

Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific ... [more ▼]

Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTC with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related pro-coagulant properties in the blood. TF blockade by antibody or shRNA diminished the pro-coagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT-TF regulatory axis which triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTC. [less ▲]

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine ... [more ▼]

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. (c)2016 AACR. [less ▲]

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to ... [more ▼]

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikke in Wnt-driven tumor development. We found that Ikke was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikke in b-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikke to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikke was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding proinflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikke-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikke phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikke to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. [less ▲]

Epithelial-mesenchymal transition (EMT) in cells is a developmental process adopted during tumorigenesis that promotes metastatic capacity. In this study, we advance understanding of EMT control in cancer ... [more ▼]

Epithelial-mesenchymal transition (EMT) in cells is a developmental process adopted during tumorigenesis that promotes metastatic capacity. In this study, we advance understanding of EMT control in cancer cells with the description of a novel vimentin-ERK axis that regulates the transcriptional activity of Slug (SNAI2). Vimentin, ERK, and Slug exhibited overlapping subcellular localization in clinical specimens of triple-negative breast carcinoma. RNAi-mediated ablation of these gene products inhibited cancer cell migration and cell invasion through a laminin-rich matrix. Biochemical analyses demonstrated direct interaction of vimentin and ERK, which promoted ERK activation and enhanced vimentin transcription. Consistent with its role as an intermediate filament, vimentin acted as a scaffold to recruit Slug to ERK and promote Slug phosphorylation at serine-87. Site-directed mutagenesis established a requirement for ERK-mediated Slug phosphorylation in EMT initiation. Together, these findings identified a pivotal step in controlling the ability of Slug to organize hallmarks of EMT. [less ▲]

MT4-MMP (MMP-17) is a GPI-anchored matrix metalloprotease expressed on the surface of cancer cells which promotes tumor growth and metastasis. In this report, we identify MT4-MMP as an important driver of ... [more ▼]

MT4-MMP (MMP-17) is a GPI-anchored matrix metalloprotease expressed on the surface of cancer cells which promotes tumor growth and metastasis. In this report, we identify MT4-MMP as an important driver of cancer cell proliferation through CDK4 activation and retinoblastoma protein (Rb) inactivation. We also determine a functional link between MT4-MMP and the growth factor receptor EGFR. Mechanistic experiments revealed direct association of MT4-MMP and its positive effects on EGFR phosphorylation in response to TGF- and EGF in cancer cells. Notably, the effects of MT4-MMP on proliferation and EGFR activation did not rely on metalloprotease activity. Clinically, MT4-MMP and EGFR expression were correlated in human triple negative breast cancer specimens. Altogether our results identify MT4-MMP as a positive modifier of EGFR outside-in signaling that acts to cooperatively drive cancer cell proliferation. [less ▲]

Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair and endocytosis. While some reports have implicated myoferlin in cancer, the extent of its ... [more ▼]

Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair and endocytosis. While some reports have implicated myoferlin in cancer, the extent of its expression in and contributions to cancer are not well established. In this study, we show that myoferlin is overexpressed in human breast cancers and that it is has a critical role in controlling degradation of the EGFR after its activation and internalization in breast cancer cells. Myoferlin depletion blocked EGF-induced cell migration and epithelial-to-mesenchymal transition. Both effects were induced as a result of impaired degradation of phosphorylated EGFR via dysfunctional plasma membrane caveolae and alteration of caveolin homooligomerization. In parallel, myoferlin depletion reduced tumor development in a chicken chorioallantoic membrane xenograft model of human breast cancer. Considering the therapeutic significance of EGFR targeting, our findings identify myoferlin as an novel candidate function to target for future drug development. [less ▲]

Estrogens directly promote the growth of breast cancers that express the Estrogen Receptor 􏰀 (ERalpha). However, the contribution of stromal expression of ERalpha in the tumor microenvironment to the pro ... [more ▼]

Estrogens directly promote the growth of breast cancers that express the Estrogen Receptor 􏰀 (ERalpha). However, the contribution of stromal expression of ERalpha in the tumor microenvironment to the pro-tumoral effects of estrogen has never been explored. In this study, we evaluated the molecular and cellular mechanisms by which 17beta-estradiol (E2) impacts the microenvironment and modulates tumor development of ERalpha-negative tumors. Using different mouse models of ER-negative cancer cells grafted subcutaneously into syngeneic ovariectomized immunocompetent mice, we found that E2 potentiates tumor growth, increases intratumoral vessel density and modifies tumor vasculature into a more regularly organized structure, thereby improving vessel stabilization to prevent tumor hypoxia and necrosis. These E2-induced effects were completely abrogated in ERalpha-deficient mice, demonstrating a critical role of host ERα. Notably, E2 did not accelerate tumor growth when ERalpha was deficient in Tie2- positive cells, but still expressed by bone marrow derived cells. These results were extended by clinical evidence of ERalpha-positive stromal cell labeling in the microenvironment of human breast cancers. Together, our findings therefore suggest that E2 promotes the growth of ERalpha-negative cancer cells through the activation of stromal ERα (not hematopoiteic but Tie2-dependent expression of ERalpha), which normalizes tumor angiogenesis and allows an adaptation of blood supply to tumor demand preventing hypoxia and necrosis. These findings significantly deepen mechanistic insights into the impact of E2 on tumor development with potential consequences for cancer treatment. [less ▲]

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRalpha) has been implicated in breast cancer and bone ... [more ▼]

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRalpha) has been implicated in breast cancer and bone development, prompting us to examine whether ERRalpha may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRalpha reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRalpha upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRalpha reduced breast cancer cell growth in bone. In contrast, ERRalpha overexpression increased breast cancer cell growth in the mammary gland. ERRalpha-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRalpha mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRalpha plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone. [less ▲]

Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the ... [more ▼]

Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the current report, we provide evidence that this activation has a more profound role. Using a mass spectrometry-based phospholipid analysis approach, we show that clinical tumor tissues that display the lipogenic phenotype show an increase in the degree of lipid saturation compared with nonlipogenic tumors. Reversal of the lipogenic switch in cancer cells by treatment with the lipogenesis inhibitor soraphen A or by targeting lipogenic enzymes with small interfering RNA leads to a marked decrease in saturated and mono-unsaturated phospholipid species and increases the relative degree of polyunsaturation. Because polyunsaturated acyl chains are more susceptible to peroxidation, inhibition of lipogenesis increases the levels of peroxidation end products and renders cells more susceptible to oxidative stress-induced cell death. As saturated lipids pack more densely, modulation of lipogenesis also alters lateral and transversal membrane dynamics as revealed by diffusion of membrane-targeted green fluorescent protein and by the uptake and response to doxorubicin. These data show that shifting lipid acquisition from lipid uptake toward de novo lipogenesis dramatically changes membrane properties and protects cells from both endogenous and exogenous insults. These findings provide important new insights into the role of de novo lipogenesis in cancer cells, and they provide a rationale for the use of lipogenesis inhibitors as antineoplastic agents and as chemotherapeutic sensitizers. [less ▲]